Human coronaviruses (H-CoVs) cause respiratory diseases ranging from self-limiting bronchiolitis to severe acute respiratory syndrome (SARS). This proposal aims to compare NL63-CoV, which causes mild disease, with SARS-CoV, focusing on distinctions between the viral spike (S) proteins that mediate the entry of these H-CoVs into host cells. The SARS and NL63 S proteins are divergent yet bind to the same host cell receptor, angiotensin converting enzyme 2 (ACE2). Subsequent to ACE2 interaction, these two S proteins may differentially direct virus entry and uncoating, in ways that may correlate with virus pathogenicity differences. Aim 1 addresses this question of distinct H-CoV entry pathways and extends preliminary data suggesting that integrins are used during NL63 S-mediated entry. Experiments will determine whether particular integrins are used by NL63 and other H-CoVs in tissue culture models of lung epithelia, and genetic approaches will be used to identify novel additional coreceptors participating in H-CoV entry. ACE2 operates as an ectopeptidase and as a signaling molecule to regulate the renin-angiotensin system and the inflammatory state of the injured lung. S proteins may disturb these central ACE2 functions. Aim 2 experiments will construct S fragments, recombinant SARS / NL63 S proteins, and HCoV-like particles bearing complete S proteins, for use as ACE2 ligands. S:ACE2 interactions leading to proinflammatory host cell responses will be identified, and the host responses to virus entry events operating subsequent to the ACE2 interaction will also be discerned. These studies relating S : ACE2 interactions with inflammation and lung pathology comprise a key collaboration with PPG component 3. The broader biological relevance of H-CoV entry cofactors and proinflammatory responses require the use of complete infectious viruses and host animal models. Robust in vitro and in vivo models to evaluate NL63-CoV are lacking. In Aim 3, we plan to select NL63-CoV variants for vigorous growth in culture, using cells overproducing ACE2 and integrins, with expectations for informative S mutants. Variants will be evaluated for growth in mice to establish models for the human disease. We will also construct recombinant NL63 / SARS chimeric viruses in collaboration with PPG component 4 to further correlate S proteins with virus growth, coreceptor usage and host cell responsiveness.